CRISPR-Cas9-based one-step multiplexed genome editing through optimizing guide RNA processing strategies in Pichia pastoris
The important methylotrophic yeast Pichia pastoris has been utilized for the production of a variety of heterologous recombinant proteins and has great potential for use in the production of value-added compounds using methanol as a substrate. However, the lack of convenient and efficient genome eng...
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KeAi Communications Co., Ltd.
2025-06-01
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| Series: | Synthetic and Systems Biotechnology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405805X25000055 |
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| author | Kaidi Chen Gulikezi Maimaitirexiati Qiannan Zhang Yi Li Xiangjian Liu Hongting Tang Xiang Gao Bo Wang Tao Yu Shuyuan Guo |
| author_facet | Kaidi Chen Gulikezi Maimaitirexiati Qiannan Zhang Yi Li Xiangjian Liu Hongting Tang Xiang Gao Bo Wang Tao Yu Shuyuan Guo |
| author_sort | Kaidi Chen |
| collection | DOAJ |
| description | The important methylotrophic yeast Pichia pastoris has been utilized for the production of a variety of heterologous recombinant proteins and has great potential for use in the production of value-added compounds using methanol as a substrate. However, the lack of convenient and efficient genome engineering tools has hindered further applications of P. pastoris, especially in complex and multistep metabolic engineering scenarios. Hence, we developed a rapid and convenient multi-gene editing system based on CRISPR/Cas9 by optimizing the guide RNA processing strategy, which can achieve dual-gene knockout or multi-gene integration in single step. Firstly, we found that the HgH (HH-sgRNA-HDV) structure achieved the highest single-gene knockout efficiency (95.8 %) among the three sgRNA processing cassettes, including a tRNA-sgRNA-tRNA (tgt) array, HgH structure and tRNA-sgRNA-HDV (tgH) structure. Furthermore, the dHgH structure (double HgH) enabled one-step dual-gene disruption and multi-gene integration. The efficiency of dual-site knockout ranged from 60 % to 100 %, with functional genes knockout achieving approximately 60 % (Δaox1Δgut1), while dual neutral sites knockout reached 100 %. Finally, we applied the system for one-step production of fatty acids and 5-hydroxytryptophan. The yield of FFAs reached 23 mg/L/μg protein/OD, while the yield of 5-hydroxytryptophan was 13.3 mg/L. The system will contribute to the application of P. pastoris as an attractive cell factory for multiplexed compound biosynthesis and will serve as a valuable tool for enhancing one-carbon (C1) bio-utilization. |
| format | Article |
| id | doaj-art-fab0014185554109b94c00ab2324e576 |
| institution | Kabale University |
| issn | 2405-805X |
| language | English |
| publishDate | 2025-06-01 |
| publisher | KeAi Communications Co., Ltd. |
| record_format | Article |
| series | Synthetic and Systems Biotechnology |
| spelling | doaj-art-fab0014185554109b94c00ab2324e5762025-02-07T04:47:52ZengKeAi Communications Co., Ltd.Synthetic and Systems Biotechnology2405-805X2025-06-01102484494CRISPR-Cas9-based one-step multiplexed genome editing through optimizing guide RNA processing strategies in Pichia pastorisKaidi Chen0Gulikezi Maimaitirexiati1Qiannan Zhang2Yi Li3Xiangjian Liu4Hongting Tang5Xiang Gao6Bo Wang7Tao Yu8Shuyuan Guo9Center for Synthetic Biochemistry, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), Shenzhen, 518055, China; University of the Chinese Academy of Sciences, Beijing, 100049, ChinaCenter for Synthetic Biochemistry, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), Shenzhen, 518055, ChinaCenter for Synthetic Biochemistry, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), Shenzhen, 518055, ChinaCenter for Synthetic Biochemistry, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), Shenzhen, 518055, China; University of the Chinese Academy of Sciences, Beijing, 100049, ChinaCenter for Synthetic Biochemistry, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), Shenzhen, 518055, ChinaSchool of Agriculture and Biotechnology, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen, 518107, ChinaCenter for Materials Synthetic Biology, CAS Key Laboratory of Quantitative Engineering Biology of CAS, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academic of Science, Shenzhen, 518055, ChinaCenter for Materials Synthetic Biology, CAS Key Laboratory of Quantitative Engineering Biology of CAS, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academic of Science, Shenzhen, 518055, ChinaCenter for Synthetic Biochemistry, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), Shenzhen, 518055, China; Corresponding author.Center for Synthetic Biochemistry, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), Shenzhen, 518055, China; Corresponding author.The important methylotrophic yeast Pichia pastoris has been utilized for the production of a variety of heterologous recombinant proteins and has great potential for use in the production of value-added compounds using methanol as a substrate. However, the lack of convenient and efficient genome engineering tools has hindered further applications of P. pastoris, especially in complex and multistep metabolic engineering scenarios. Hence, we developed a rapid and convenient multi-gene editing system based on CRISPR/Cas9 by optimizing the guide RNA processing strategy, which can achieve dual-gene knockout or multi-gene integration in single step. Firstly, we found that the HgH (HH-sgRNA-HDV) structure achieved the highest single-gene knockout efficiency (95.8 %) among the three sgRNA processing cassettes, including a tRNA-sgRNA-tRNA (tgt) array, HgH structure and tRNA-sgRNA-HDV (tgH) structure. Furthermore, the dHgH structure (double HgH) enabled one-step dual-gene disruption and multi-gene integration. The efficiency of dual-site knockout ranged from 60 % to 100 %, with functional genes knockout achieving approximately 60 % (Δaox1Δgut1), while dual neutral sites knockout reached 100 %. Finally, we applied the system for one-step production of fatty acids and 5-hydroxytryptophan. The yield of FFAs reached 23 mg/L/μg protein/OD, while the yield of 5-hydroxytryptophan was 13.3 mg/L. The system will contribute to the application of P. pastoris as an attractive cell factory for multiplexed compound biosynthesis and will serve as a valuable tool for enhancing one-carbon (C1) bio-utilization.http://www.sciencedirect.com/science/article/pii/S2405805X25000055Pichia pastorisGene deletion and integrationCRISPR/Cas9Genome editinggRNA processing |
| spellingShingle | Kaidi Chen Gulikezi Maimaitirexiati Qiannan Zhang Yi Li Xiangjian Liu Hongting Tang Xiang Gao Bo Wang Tao Yu Shuyuan Guo CRISPR-Cas9-based one-step multiplexed genome editing through optimizing guide RNA processing strategies in Pichia pastoris Synthetic and Systems Biotechnology Pichia pastoris Gene deletion and integration CRISPR/Cas9 Genome editing gRNA processing |
| title | CRISPR-Cas9-based one-step multiplexed genome editing through optimizing guide RNA processing strategies in Pichia pastoris |
| title_full | CRISPR-Cas9-based one-step multiplexed genome editing through optimizing guide RNA processing strategies in Pichia pastoris |
| title_fullStr | CRISPR-Cas9-based one-step multiplexed genome editing through optimizing guide RNA processing strategies in Pichia pastoris |
| title_full_unstemmed | CRISPR-Cas9-based one-step multiplexed genome editing through optimizing guide RNA processing strategies in Pichia pastoris |
| title_short | CRISPR-Cas9-based one-step multiplexed genome editing through optimizing guide RNA processing strategies in Pichia pastoris |
| title_sort | crispr cas9 based one step multiplexed genome editing through optimizing guide rna processing strategies in pichia pastoris |
| topic | Pichia pastoris Gene deletion and integration CRISPR/Cas9 Genome editing gRNA processing |
| url | http://www.sciencedirect.com/science/article/pii/S2405805X25000055 |
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